Cells in most tissues are interconnected by cell-cell channels which allow the passage of electrolytes and small molecules from cell to cell. Aggregates of these channels are contained in the clustered intramembranous particles of gap junctions. In excitable cells these channels are instrumental for impulse propagation, in other tissues they are thought to pass signal molecules and to synchronize the cells metabolically. The objective of this research project is to understand the process of formation of cell-cell channels. This problem is addressed by functional expression of cell-cell channels in paired oocytes from cloned gap junction (connexin) cDNA. The oocyte assay will be used in combination with mutagenesis, site-specific reagents, tracer flux measurements, and the patch clamp technique to obtain information about molecular domains involved in various aspects of the channel formation process like the docking of the extracellular domains to each other and the associated docking gate. The pore lining will be assayed with a movable probe, using the cysteine scanning mutagenesis approach. Another aspect of this research project deals with the inhibition of gap junction channel formation by the testis-specific connexin33. Using the oocyte assay the mechanism of inhibition is to be investigated with the expectation to obtain clues about the channel formation process. Using transgenic mice the physiological role of cx33 inhibition will be studied. The mice will carry reporter genes for cx33 or gene constructs where the cx33 promoter drives the expression of a connexin (cx32) that is uninhibited by cx33. Expression of cx32 under the control of the cx33 promoter is expected to override the inhibitory role of cx33 and the ensuing phenotypic changes should reveal the physiological role of cx33 inhibition.